Project Abstract/Summary: Insulin degrading enzyme (IDE), an evolutionarily conserved metalloprotease, is involved in the clearance of structurally diverse, bioactive peptides, e.g. three glucose regulating hormones (insulin, amylin, and glucagon), amyloid ? (A?), and CC chemokines. Accumulating evidence strongly supports the role of IDE in the progression of several chronic human diseases including type 2 Diabetes Mellitus and Alzheimer's disease. We have solved the structures of human IDE in the presence and absence of diverse substrates. In conjunction with our biophysical and biochemical analyses, our studies reveal the molecular basis of how IDE uses an enclosed catalytic chamber to selectively recognize the tertiary structural features of their substrates to unfold and degrade them. Our long-term goals are to elucidate the selective recognition of toxic peptides by IDE in proteostasis, delineate the normal and pathologic functions of IDE, and develop small molecules that modulate IDE activity for treatment of various human diseases such as type 2 diabetes and Alzheimer disease. Our objectives for this application are to gain structural insights into the molecular dynamics of IDE and how the structural dynamics of IDE are linked to their ability to recognize amyloid peptides. In addition, we will develop potent chemical modulators of IDE to probe and modulate the biological functions of IDE. Our research rationale is that understanding the regulation and functions of IDE as well as the development of small chemical modulators of IDE will ultimately allow us to better design IDE-based therapy for the treatment of certain human diseases such as diabetes, Alzheimer's disease, and inflammation. Three specific aims are proposed. In Aim 1, we will integrate X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, cryo-electron microscopy, small angle X-ray scattering, and molecular dynamic simulation to address the conformational switches governing how IDE selectively degrades amyloid peptides that are diverse in size and shape. Aim 2 is to analyze the link between IDE with type 2 diabetes using small molecule inhibitors and IDE variants in type 2 diabetes patients. For Aim 3, we will discover small molecule activators or activating mutations of IDE to enhance A? clearance as well as analyze the role of IDE in the clearance of CCL3 and CCL4 in vivo. This work is significant because it will define structures of multiple IDE open states, which are the key IDE conformational states for substrate recognition. Further, this work will provide novel small molecule probes for manipulating IDE activity in vivo. Finally, the studies will investigate the functional roles of IDE in human diseases. This work is innovative because novel integrative structural approaches will be explored to examine the conformational changes of IDE and novel small molecule discovery methods will be applied to identify new small molecule IDE inhibitors and activators.